- Dilute 30μg of total protein with lysis buffer to 15μl of total
volume, mixed with 5μlof4loading buffer, and heat at
95 C for 5 min. Afterward the samples can be stored at
20 C until further analysis by SDS-PAGE (seeSubheading
3.5)(seeNote 27).
3.5 SDS
Polyacrylamide Gel
Electrophoresis
and Immunoblotting
- For a 7.5% SDS polyacrylamide gel, mix 5 ml of H 2 O, 2.5 ml of
polyacrylamide solution, and 2.5 ml of resolving gel buffer (see
Note 28). For a 15% SDS polyacrylamide gel, mix 2.5 ml of
H 2 O, 5 ml of polyacrylamide solution, and 2.5 ml of resolving
gel buffer (seeNote 29). Start polymerization by adding 65μl
of APS and 15μl of TEMED. - The 5% stacking gel consists of 3.125 ml of H 2 O, 1.25 ml of
stacking gel buffer, and 0.625 ml of polyacrylamide solution.
To start the polymerization, add 25μl of APS and 8μlof
TEMED. - Load the samples from Subheading3.4 onto a 7.5% or a 15%
SDS gel and separate electrophoretically at 150 V for about
1.5 h until the bromophenol dye front just ran off the gel (see
Notes 3and 27 ). - Blot the 15% SDS gels with the semidry method. Equilibrate
six Whatman filter papers, a nitrocellulose membrane, and the
SDS gel for a few minutes with semidry transfer buffer. After-
ward stack them on the semidry blotting apparatus in the
following order starting from the cathode: three filter papers,
SDS gel, nitrocellulose membrane, and three filter papers.
Perform the transfer at 1.9 mA per cm^2 of nitrocellulose mem-
brane for 1.25 h and maximal 40 V. - Blot the 7.5% SDS gels with the wet blotting method. Equili-
brate two sponges, six Whatman filter papers, a nitrocellulose
membrane, and the SDS gel in ice-cold tank blot transfer buffer
for a few minutes. Afterward stack the holder cassette inside the
container with the black side facing down in the following
order: a sponge, three filter papers, SDS gel, nitrocellulose
membrane, three filter papers, and a sponge. To remove bub-
bles from the stack, use a glass pipette and roll over it carefully.
Close the holder cassette carefully in the container, and place
into the blotting chamber with the black side facing black and
the white side facing red. Place the cooling unit inside the
chamber as well, and fill it up with ice-cold tank blot transfer
buffer. Run the tank blot at 100 V for 1 h. - After the transfer, incubate the blots with IB blocking solution
for at least 1 h at room temperature on a horizontal shaker. - Subsequently wash the blocked blots several times in TBS-T
(seeNote 30) until the incubation with primary antibody at
4 C overnight on a rocking device (seeNote 2).
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